Evaluation of suitable reference genes for gene expression analysis in the northern root-knot nematode, Meloidogyne hapla.

Abstract

The northern root-knot nematode (Meloidogyne hapla) is a critical pathogen with a wide host range. Quantitative real-time polymerase chain reaction (qRT-PCR) has been used to elucidate gene expression and function of M. hapla. Suitable reference genes are required to ensure accurate results of qRT-PCR for normalising gene expression. Eleven candidate reference genes of M. hapla were selected to evaluate gene expression stability under different conditions. The stability of candidate reference genes was ranked using RefFinder analysis, and the optimal number of reference genes was recommended with geNorm. Notably, the most stable reference genes were SDHA, Mdh, and RpS6 for all samples; SDHA and RpS6 were particularly stable during development stage treatments, whereas Mdh and RpS6 were appropriate for temperature and inorganic compound treatments. In contrast, the least stable reference genes were Actin1 during development stages and all other treatments, GAPDH for temperature treatments, and α-Tub for inorganic compound treatments. One target gene, Mh-Hsp90, was used to verify the selection of reference genes, results showed Mdh and RpS6 could be used as suitable reference genes for M. hapla, and Mdh plus RpS6 were better. Our finding contributes to further work on gene transcription analysis in M. hapla.